The intracellular lifecycle of Mycobacterium tuberculosis is completely unexplored. Our understanding of the physiology of the tubercle bacillus is limited by our artificial in vitro axenic growth conditions. The completion of the genomic DNA sequence of a virulent strain of the bacterium (H37Rv) offered a glimpse into the varied metabolic potential of this complex Actinomycete. Far from the textbook description of an obligate aerobe, the tubercle bacillus has preserved all of the machinery necessary for life without oxygen. Likewise, far from growth on glucose in a laboratory flask the bacillus appears to be supremely adapted for living off of abundant host lipid constituents. The studies encompassed within this project all have in common the aim of discerning the nature of the intracellular physiology of Mycobacterium tuberculosis during the process of human disease. These studies have been performed and facilitated the availability of the genome sequence. One set of these studies involves an exploration of a large gene locus we predicted in the genome paper would be sufficient to encode the necessary information to produce the small, peptide-derived iron acquiring molecules of M. tuberculosis. By constructing directed knockouts we were able to confirm this genomic prediction and demonstrate conclusively that these molecules are required specifically for growth under the iron-limiting conditions of the mammalian cell. In another set of studies we used a genomic synthesis of regulatory pathways to predict that a small hyperphosphorylated nucleotide would coordinate entry of the bacteria into stationary phase. We predicted that disabling this regulatory network would eliminate the organisms' ability to enter dormancy and interfere with long-term survival. Again the ability to genetically manipulate the bacteria allowed us to test this prediction and establish that the mutant bacteria were disabled for long-term survival without continuous growth. Finally, the suggestion from the genome that potent immunoregulatory molecules called polyketides were produced led us to explore the immunomodulatory effects of various mycobacterial lipidic fractions from recent patient isolates in collaboration with a group at Rockefeller University.